Karyological Data Research Articles

Experience in the production and clinical application of the cell-based medicinal product Easytense® for the repair of cartilage defects of the human knee

INTRODUCTION. The current cell-based cartilage repair methods, such as autologous chondrocyte transplantation, are not sufficiently effective, and the surgery is painful and traumatic. Therefore, there is a need for a more effective cell therapy product with a minimally invasive surgical procedure for its implantation into the patient.AIM. This study aimed to develop a manufacturing technology for the production of an autologous cell-based medicinal product (CBMP) comprising three-dimensional structures (3D-spheroids) based on chondrocytes isolated from the patient’s cartilage tissue, as well as to evaluate its clinical efficacy.MATERIALS AND METHODS. Autologous chondrocytes isolated from the patient’s cartilage biopsy were propagated in monolayer culture to obtain the required number of cells. Subsequently, the chondrocytes were cultivated on plates with a non-adhesive coating to form 3D spheroids. All CBMP production steps were performed under aseptic conditions in cell culture isolators. The authors used phase-contrast microscopy and immunohistochemical staining with specific fluorescence-labelled antibodies to characterise chondrocyte phenotypes at different stages of cultivation. Genetic stability was controlled by karyotyping. The efficacy of Easytense® was evaluated in a clinical trial using specialised functional tests and the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score. The primary efficacy endpoint was a change in the overall score on the Knee Injury and Osteoarthritis Outcome Score (KOOS).RESULTS. A manufacturing technology without using animal sera, growth factors, cytokines, or other additives was developed for the production of the autologous CBMP Easytense®. Karyological data confirmed that the chondrocytes retained genetic stability for 3 passages in monolayer culture. When cultured as 3D spheroids, the chondrocytes produced cartilage extracellular matrix proteins (type II collagen, aggrecan), thus acquiring the ability to repair damaged cartilage. The clinical trial demonstrated a statistically significant improvement in knee cartilage 12 months after the transplantation of 3D spheroids derived from autologous chondrocytes. The mean change in the overall KOOS score was 23.8±15.9.CONCLUSIONS. The clinical trial results indicate that Easytense® is highly effective for cartilage repair. Based on these results, the CBMP has been granted marketing authorisation and introduced into clinical practice in the Russian Federation. Easytense® has the potential to replace endoprosthetics and expensive surgeries abroad.

Estudos anatômicos, cariotípicos e de conteúdo de DNA nuclear em quatro morfotipos de alface silvestre

ABSTRACT Wild lettuce (Lactuca aff. canadensis L.), belonging to the Asteraceae family, occurs subspontaneously in Brazil and may originate from Africa, Asia, Europe and North America. Popularly known as Canada lettuce, it is an unconventional leafy vegetable. Studies on this species are scarce in Brazil, and its scientific name is debated among experts. It has high morphological variability and controversial botanical classification. This study characterized the stomata, organized the karyotype, and determined the nuclear DNA content of four morphotypes of wild lettuce to facilitate correct classification. The genetic material used was acquired from the non-conventional vegetable germplasm in UFLA. The leaves of wild lettuce morphotypes are hypoamphiestomatic with a greater number of stomata in the abaxial epidermis. There were similarities between the morphotypes (green and purple) and between the smooth purple types (narrow leaf and broad leaf) for the number and size of stomata. No variation was found in the number of chromosomes (2n = 18) or DNA content among the four morphotypes. The separation of the morphotypes of wild lettuce did not match the morphological classification or the karyological data. The four morphotypes evaluated were placed under the same species, and the results obtained when compared to other studies led us to infer that the four morphotypes of wild Lactuca belonged to the species L. indica and not L. canadensis as previously assumed. Further investigation may provide insights into the evolutionary history of this species.

Cytological Studies on Some Members of Commelinaceae Mirb. from Kangra Valley (Himach al Pradesh ) With a Short Summary of Karyological Data on the Analyzed Genera

Cytological Studies on Some Members of Commelinaceae Mirb. from Kangra Valley (Himach al Pradesh ) With a Short Summary of Karyological Data on the Analyzed Genera

Systematics of the genus Euryoryzomys Weksler et al., 2006 (Rodentia: Cricetidae): integrative analysis reveals a new species from north-eastern Brazil

Abstract The genus Euryoryzomys encompasses six species found across Cis-Andean lowland and montane rainforests in South America. One of the most enigmatic forms of the genus is found in the Brejos de altitude of north-eastern Brazil, where enclaves of dense rainforest in mountain ranges sharply contrast with surrounding semi-arid Caatinga. Here we analyse morphological and molecular data, and demonstrate that these populations are members of a new species, which is described and contrasted with other Euryoryzomys. Phylogenetic analyses based on mitochondrial cytochrome b gene, coupled with morphometrics and comparative anatomy analyses, show that the new species differs from its sister-species, Euryoryzomys lamia from the Cerrado of Central Brazil, and from other forms of the genus, corroborating previous perspectives based on karyological data. The new species of the genus occurs in two distinct areas in the state of Ceará, Serra de Ibiapaba and Serra de Baturité, and one isolated range in Paraíba state. The recovered biogeographic pattern of Brejos de altitude—Cerrado sister-group relationship is novel among studied vertebrates, as most species from Brejos are more closely related to taxa from Amazon and Atlantic Forest. Further taxonomic efforts are necessary to uncover the total diversity within Euryoryzomys, especially for the nominal taxa E. emmonsae, E. macconnelli, and E. nitidus.

Karyotype diversity in four Terminalia L. species and its taxonomic implications

AbstractFour Terminalia L. species namely, T. arjuna (Roxb.) Wight and Arn., T. bellirica (Gaertn.) Roxb., T. chebula Retz. and T. catappa L. were studied to evaluate the chromosomal relationship corresponding with their taxonomic characters. Analysis of mitotic metaphases showed that the somatic chromosome numbers and the centromeric formulas of these species were 2n = 24 (16m + 8sm) for T. arjuna, 2n = 48 (36m + 12sm) for T. bellirica, 2n = 48 (46m + 2sm) for T. chebula and 2n = 24 (24m) for T. catappa. Two pairs of satellites were found in T. bellirica and T. catappa. Based on different karyological data, T. chebula was positioned in 1A, T. arjuna and T. catappa in 1B and T. bellirica in 2B. Based on comparative karyomorphological data, T. bellirica and T. arjuna presented more advanced features in comparison with T. chebula and T. catappa, which also correlated with the taxonomic characters in respect of the evolutionary point of view. Therefore, the present investigation effectively evaluated chromosomal relationships with morphological implications that conferred upon evolutionary changes among these four Terminalia species.

Molecular phylogeny of the snorkel snail Rhiostoma housei, a species complex from Thailand with descriptions of three new species

Snorkel snails (genus Rhiostoma) are widely distributed in Indo-China and on the Malay Peninsula. The shell morphology is traditionally used for species identification yet in Thailand, the common snorkel snail, Rhiostoma housei, shows considerable variation in shell morphology within and between populations. Therefore species identifications and delimitations are difficult. We used two mitochondrial DNA fragments (COI and 16S rRNA) and morphological characters to delimit species and infer phylogenetic relationships of Rhiostoma housei s.l. from eight localities in Thailand, representing potential cryptic species suggested by earlier allozyme and karyological data. Results revealed four distinct clades from different geographic areas in Thailand. Species delimitation analyses confirmed the clades as four separate species and a geometric morphometric analysis demonstrated subtle but consistent conchological differences between the four clades. The high COI sequence divergences among the four clades (mean: 14.8%; range: 10.3–16.5%) further supported the species level recognition. As a consequence, three new species are described from Thailand: R. khoratense, sp. nov., R. nakwangense, sp. nov. and R. phunangense, sp. nov. ZooBank: urn:lsid:zoobank.org:pub:EE534749-9346-4972-BF2E-3B5D0EB8AED8

Centaurea dolopica (Asteraceae, Cardueae), a new species of section Acrocentron from Greece

Centaurea dolopica from Omvriaki area in Central Greece is described and illustrated as a species new to science based on morphological and molecular evidence. Detailed description of the new species, distribution, ecology, conservation status, as well as comparison with closely related species are provided. Individuals of Centaurea dolopica were also examined karyologically resulting to the chromosome number 2n = 2x = 22 accompanied by comprehensive karyotype analysis. Phylogenetic study including the new species and major representatives of Centaurea section Acrocentron was conducted using two nuclear markers (ITS, ETS). The results revealed the placement of Centaurea dolopica in the Greek-Aegean clade of the section in close evolutionary relationship with the members of the C. achaia group, corroborating the morphological and karyological data.

DCDB: Chromosome Database of Tribe Delphinieae (Ranunculaceae): Structure, Exploitation, and Recent Development.

An updated (and now online) version of the former chromosome database of tribe Delphinieae (Ranunculaceae) is presented ( http://www.delphinieae.online ). This new version is the result of an accurate, exhaustive literature and Internet research, by adding chromosome counts and all related karyological information for the genera Aconitum L., Gymnaconitum (Stapf) Wei Wang & Z. D. Chen, Delphinium L. (including Staphisagria Spach), Consolida (DC.) S. F. Gray and Aconitella Spach, accumulated during the approximately last 25years and that comprise worldwide published data from 1889 to 2021. The Delphinieae Chromosome Database (DCDB) (last updated 31.12.2021) contains a total number of 3435 reports belonging to 425 species (503 taxa), which represents 48.6% of the total species of the tribe (an increase of c. 213% and 32% compared with the 1097 and 2598 reports gathered in the 1999 and 2016 versions, respectively). This increase is due both to chromosome research progress and to improved information capture system. Moreover, recent taxonomic advances, synonymization, and new phylogenetic criteria have also been considered.The DCDB database provides the most complete currently available information on published chromosome numbers, ploidy-level estimates, and other karyological data of Delphinieae, and it is aimed to be useful for the building of cytotaxonomical databases and for specific research ongoing projects of systematics and evolution of Ranunculaceae. DCDB includes two levels of taxonomic resolution, published name (original and standardized form) and database accepted name (based on either Plants of the World - POWO or expert specialist criteria), as well as the geographic origin of each count (country, locality, geographic coordinates, elevation, or ecological information when reported in the original publication), associated karyological data if originally provided (studied material type, graphic information, chromosome measures and formulas, satellites, B chromosomes, other cytogenetic techniques used, etc.), voucher information and reference (with DOI and other links to access the original document). An effort to check the original sources and to search in grey literature allowed to indicate the counts that appear to be registered twice.

CromoCat: Chromosome Database of the Vascular Flora of the Catalan Countries-25 years.

CromoCat is a plant chromosome database that evolved from previous versions, as a repository of karyological information on the vascular flora of the Catalan Countries. CromoCat is designed as an independent database, managed by a team based at the University of Barcelona directed by J. Simon, available from its own webpage ( http://www.cromo.cat/ )and from the Flora section of the Catalan Biodiversity Database - BDBC ( http://biodiver.bio.ub.es ). CromoCat contains at present (mid 2022) more than 68,000 records of karyological data belonging to more than 5000 taxa. A synthesis of the development of CromoCat, its functional system, achievements, limitations, and adopted solutions, during 25years (1996-2021) and updated 2022, as well as the application to biodiversity conservation and management are outlined.

Karyological data of two Sicilian endemic Centaurea species

Karyological data of two Sicilian endemic Centaurea species

Karyological data of some Greek taxa

Karyological data of some Greek taxa

Leaf and stem anatomy and micromorphology of four Inuleae (Compositae) genera, with notes on their taxonomic significance

We studied the leaf and stem anatomy and micromorphology of 17 Inula, Dittrichia, Limbarda and Pulicaria taxa native to the Balkan region, aiming to identify additional characters that may help to define the taxa within this group.The taxa analysed were all previously placed in different sections within Inula, but have later been segregated into separate genera. According to the latest molecular phylogenetic analyses, as well as morphological and karyological data, Inula sect. Corvisartia, including the type species of the genus (I. helenium), should be segregated from the remaining Inula species as a separate genus. However, data on the anatomical and micromorphological structure of the vegetative organs of the analyzed taxa have been sparse and incomplete, and previous analyses were carried out on a small number of taxa, with less emphasis on comparisons. We studied more than 50 qualitative and quantitative characters that were analyzed and measured using dissecting, compound and scanning electron microscopes and the data were statistically processed. The results show that certain characters are useful for separating individual taxa, but their grouping does not follow the current taxonomic classification. However, I. helenium is clearly distinguished from the remaining taxa, which supports its separation as a separate genus.

New Chromosomal Data of Dianthus Section Leiopetali (Caryophyllaceae, Sileneae)

In section Leiopetali, the diploid chromosome numbers were reported from only six of total 25 taxa. There is no karyological data relating to other 19 taxa. In this study, it is intended to determine the diploid chromosome numbers of taxa of section Leiopetali. The diploid number of 13 species were detected, four of which were recorded for the first time and nine numbers compatible with the previous reports. Twelve species were diploid with 2n = 2x =30. D. leptopetalus was diploid and polyploid, which reveals only one polyploidy level of tetraploidy (2n = 4x = 60). Polyploidy might have played a role in the karyotype evolution of the genus. One of the most important reasons for this situation is that although polyploidy is observed in the genus Dianthus, the dysploidy mechanism that causes a change in the basic chromosome number has not been found until now. The basic number is x = 15 according to the all chromosomal reports. In conclusion, this study reports new data into the karyological characteristics of section Leiopetali (genus Dianthus) that can be useful for interpreting or understanding relationships among sections.

Polysomaty and chromosome number variation in a population of Ceratophyllum demersum L. from Aquila Lake (Aspromonte Mountains, Calabria, Italy)

Ceratophyllum is a genus of perennial, freshwater plants with an essentially cosmopolitan distribution. In this study, we report for the first time, the presence of somatic polyploidy and chromosome number variation in a C. demersum population from Calabria (Italy). Observations on mitotic divisions and chromosomal behaviors as well as DNA measurements have been carried out using standard chromosome counting and Image Cytometric analysis. The estimations of DNA amounts were carried out using the interphase-peak method. Chromosome number variation was detected in different plants from the studied population, as well as in a single individual. The relative DNA value of nuclei confirmed that all the studied individuals presented polysomaty. Anomalies during the different mitotic phases were also observed. The data strongly suggest that these anomalies associated to the endoreduplication could be the cause of the several numbers detected. An attempt to compare our data with those reported in literature is discussed. However, the detection in Calabrian population of different chromosome numbers, even in a single individual, strongly suggests that a revision of karyological data in this genus seems necessary. The data reported represent the first contribution to understanding the basic cytogenetics in this genus.

Review of song patterns and sound production in armoured ground crickets (Orthoptera: Tettigoniidae: Hetrodini) with karyological data and taxonomic notes.

In the mate finding system of bush-crickets, acoustical signals play a central role. Here we review and describe the bioacoustics of Hetrodini, a morphologically uniform group of Tettigonioidea with a distribution centered in Africa. The male calling songs are produced by tegmino-tegminal stridulation. In all species, these fore wings are completely covered by the pronotum (invisible in intact specimens) and lack the glossy mirror cells which are well-known from many other singing Ensifera. Concerning spectral composition, the broad frequency peak and the parts with the most energy lie in the high audio or low ultrasonic range. In amplitude modulation, the songs are relatively simply structured and contain only one type of syllables each. These syllables consist of heavily dampened impulses (non-resonant song) and are arranged in long series of echemes (chirps) or trills. As far as it is known, syntopic species differ in syllable and/or echeme repetition rates. In contrast to the uniform morphology, the karyotypes are surprisingly diverse with chromosome numbers ranging from 2n = 29 to 17 and one of two sex determination systems: X0 and neo-XY. In our opinion, taxonomically the group is at the moment at best considered as a tribe incertae sedis (without subfamily classification) within Tettigoniidae (not Tettigoniinae sensu OSF).

Comparative chromosomal features and new karyological data in Salvia: B-chromosomes, polyploidy, dysploidy and symmetric karyotypes

Comparative chromosomal features and new karyological data in Salvia: B-chromosomes, polyploidy, dysploidy and symmetric karyotypes

New Chromosome Numbers and Karyotype Analysis of Tordylium (Apiaceae) Species with Emphasis on Their Systematics and Evolutionary Significance

In Turkey, there are eighteen Tordylium (Apiaceae) species out of twenty in the worldwide and seven of them are endemic. In this research, karyotypes of T. apulum, T. pestalozzae, T. syriacum, T. trachycarpum and karyotypes and chromosome numbers of T. cappadocicum, T. aegaeum, T. hasselquistiae, T. ketenoglui (endemic), T. macropetalum (endemic), T. pustulosum (endemic) are provided for the first time. Tordylium species were separated into five cytotypes according to chromosome numbers as 2n=8, 16, 18, 20 and 22. Besides, idiograms of haploid chromosome set, length of the chromosome arms, arm ratios, centromeric index, relative length and haploid karyotype formulas are given. T. ketenoglui has longest chromosome lengths (3,39–1,66 µm) while T. aegaeum has shortest (1,10–0,59 µm). Additionally, principal component analysis (PCA) was performed by quantitified karyological characters. PCA results showed that the median type and haploid chromosome length have significant variants among karyological characters. Also, it is discussed correlation among karyological data, current infrageneric classification and the phylogenetic hypothesis based morphological dataset of Tordylium. Karyological characters are incompatible with current infrageneric classification, similarly with conflict between the phylogenetic hypothesis and infrageneric classification. In the light of the phylogeny, it can be said that the ancestral number is 2n=16.

Comparative cytogenetics on eight Malagasy Mantellinae (Anura, Mantellidae) and a synthesis of the karyological data on the subfamily.

We performed a molecular and cytogenetic analysis on different Mantellinae species and revised the available chromosomal data on this group to provide an updated assessment of its karyological diversity and evolution. Using a fragment of the mitochondrial 16S rRNA, we performed a molecular taxonomic identification of the samples that were used for cytogenetic analyses. A comparative cytogenetic analysis, with Giemsa’s staining, Ag-NOR staining and sequential C-banding + Giemsa + CMA + DAPI was performed on eight species: Gephyromantis sp. Ca19, G.striatus (Vences, Glaw, Andreone, Jesu et Schimmenti, 2002), Mantidactylus (Chonomantis) sp. Ca11, M. (Brygoomantis) alutus (Peracca, 1893), M. (Hylobatrachus) cowanii (Boulenger, 1882), Spinomantispropeaglavei “North” (Methuen et Hewitt, 1913), S.phantasticus (Glaw et Vences, 1997) and S. sp. Ca3. Gephyromantisstriatus, M. (Brygoomantis) alutus and Spinomantispropeaglavei “North” have a karyotype of 2n = 24 chromosomes while the other species show 2n = 26 chromosomes. Among the analysed species we detected differences in the number and position of telocentric elements, location of NOR loci (alternatively on the 6th, 7th or 10th pair) and in the distribution of heterochromatin, which shows species-specific patterns. Merging our data with those previously available, we propose a karyotype of 2n = 26 with all biarmed elements and loci of NORs on the 6th chromosome pair as the ancestral state in the whole family Mantellidae. From this putative ancestral condition, a reduction of chromosome number through similar tandem fusions (from 2n = 26 to 2n = 24) occurred independently in Mantidactylus Boulenger, 1895 (subgenus Brygoomantis Dubois, 1992), Spinomantis Dubois, 1992 and Gephyromantis Methuen, 1920. Similarly, a relocation of NORs, from the putative primitive configuration on the 6th chromosome, occurred independently in Gephyromantis, Blommersia Dubois, 1992, Guibemantis Dubois, 1992, Mantella Boulenger, 1882 and Spinomantis. Chromosome inversions of primitive biarmed elements likely generated a variable number of telocentric elements in Mantellanigricans Guibé, 1978 and a different number of taxa of Gephyromantis (subgenera Duboimantis Glaw et Vences, 2006 and Laurentomantis Dubois, 1980) and Mantidactylus (subgenera Brygoomantis, Chonomantis Glaw et Vences, 1994, Hylobatrachus Laurent, 1943 and Ochthomantis Glaw et Vences, 1994).

The morphological, karyological and phylogenetic analyses of three Artemisia L. (Asteraceae) species that around the Van Lake in Turkey

Artemisia is one of the biggest genera in the family Asteraceae, with around 500-600 taxa at specific and sub-specific levels and organised in five subgenera. Due to the high number of taxa, a lot taxonomists are trying to solve the problem of its classification and phylogeny but its natural classification still has not been achieved. The aim of this study is to try to solve the problematic systematic relationship between three different Artemisia species growing in close proximity to each other in the light of morphological, karyological and molecular data. The roots, stems, leaves, flowers structures of the plant samples collected from different populations belong to these species were investigated within the framework of morphological studies. Additionally, the chromosome counts and karyotype analysises of these species were made and idiograms were drawn in the karyological studies. In the context of phylogenetic studies, ITS (ITS1-5.8S-ITS2) and trnT - trnL3’ regions of 22 individuals belonging to 3 taxa were studied. According to results of phylogenetic anlysis, it has been found that there is completed speciation genetic isolation mechanism between the species Artemisia spicigera, Artemisia taurica and Artemisia fragrans that inhibit gene flow. Also Artemisia fragrans and Artemisia spicigera species are very similar to each other in terms of morphological characteristics. However, since populations of the species Artemisia fragrans are autopolyploid, the dimensional values of their morphological squares are larger than those of the species Artemisia spicigera. This study is important as it is the first molecular based study relating with some species of Artemisia growing naturally in Turkey.

ПОЛИПЛОИДИЯ У ВОДНЫХ И ПРИБРЕЖНО-ВОДНЫХ СОСУДИСТЫХ РАСТЕНИЙ СЕВЕРО-ВОСТОКА АЗИИ

The chromosome numbers of aquatic and semiaquatic vascular plants growing in extreme North-East Asia, east of the Lena River, were analyzed. We have reviewed the information about the karyological peculiarity of the aquatic flora based on published data, including our own definitions. The karyological data are considered depending on the taxonomic position, distribution and ecological characteristics of the species. The chromosome numbers are known for 119 out of 123 species. There is no data on Zannichellia komarovii, Eleocharis termale, Stuckenia subretusa and Potamogeton sibiricus. In the aquatic flora of North-East Asia, the proportion of polyploids is higher (79%) than that reported for the entire Beringian flora (69%). We analyzed species for the presence of polyploidy depending on the type of area. Analysis by latitudinal groups revealed that all Arctic and hypoarctic species of aquatic plants are polyploids. The minimum proportion of polyploids is observed among arctoboreal species. Analysis by longitudinal groups revealed that the lowest proportion of polyploids was noted among species found only in Eurasia and absent in North America (61%), and it was the highest among pluriregional species (96%). The species of the families Cyperaceae, Lemnaceae, Ranunculaceae and Poaceae are characterized by a high level of variability in the number of chromosomes. The greatest variety of chromosome numbers is observed in the polymorphic species Caltha palustris s. l., Agrostis stolonifera, Dupontia fisheri s. l., Phragmites australis, in all species of the genus Eleocharis, and in Nymphaea tetragona, Acorus calamus, Calla palustris. The isolated cases of a high level of ploidy were found in the extreme northeast, at the northern borders of ranges, in 3 species - Hippuris vulgaris, Arctophila fulva and Beckmannia syzigachne. Only diploids are known in the families Equisetaceae, Typhaceae, Zosteraceae from the flora of North East Asia. We revealed the existence of several chromosomal races in Ranunculus gmelinii and Caltha palustris (Ranunculaceae) in the Magadan region; the distribution of chromosomal races is partly related to geographic location and partly to plant habitats. Rare karyotypes in these species are observed in aquatic forms with mainly vegetative reproduction, growing in non-freezing watercourses.